Diphenylhexatriene Fluorescence Research Articles

Coexistence of a Two-States Organization for a Cell-Penetrating Peptide in Lipid Bilayer

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P ( α) , a primary amphipathic cell-penetrating peptide which remains α-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P ( α) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P ( α) only exerted very limited effects on the corresponding liposome’s bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P ( α) . The simultaneous two-states organization of P ( α) , at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

Mitochondrial Lipid Abnormality and Electron Transport Chain Impairment in Mice Lacking α-Synuclein

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.

A new method for measuring fibrillar amyloid β‐protein using diphenylhexatriene, a fluorescent probe

The two current methods for the measurement of fibrillar (f) amyloid beta‐protein (Aβ), namely Thioflavin T (ThT) fluorescence spectroscopy and Congo red (CR) absorbance spectroscopy, are based on the interaction of fAβ with cationic ThT and anionic CR molecules. The presence of cations and colored factors in the reaction often interferes with ThT and CR assays. Recently, we reported that fibrillar Aβ forms a membrane‐like hydrophobic domain (Chauhan et al. 2001; Neuroreport12, 587–590). We report here that diphenylhexatriene (DPH), a neutral fluorescent probe, interacts with fibrillar Aβ 1‐40 and fibrillar Aβ 1‐42, but not with soluble Aβ. As a result, DPH fluorescence increases several fold in the presence of fibrillar Aβ at the excitation and emission wavelength of 360 and 430 nm, respectively. The blank sample (DPH in buffer) had minimal fluorescence, and soluble Aβ had no effect on DPH fluorescence. The interaction of DPH with fibrillar Aβ was dependent on the incubation time of these two constituents. At fixed Aβ concentration and varying DPH concentration, there was a lag phase of DPH fluorescence from 1 to 4 μm DPH, that was followed by a linear increase in fluorescence from 4 to 8 μm DPH which then reached a plateau. Studies at varying pH showed that emission spectra of fAβ/DPH can be studied at a wide range of pH from 4 to 8. Aβ concentration studies showed that the fluorescence of DPH increases linearly with increase in the concentration of fibrillar Aβ 1‐40/Aβ 1‐42. Kinetic studies showed that one mole of fAβ binds to 15.5 mol of DPH. These results suggest that DPH fluorescence can be used as a standard assay method for the measurement of fibrillar Aβ.

Abnormalities in lipid composition of brush-border membranes isolated from renal cortex of spontaneously hypertensive rats

The lipid composition and fluidity in brush-border membrane vesicles (BBMV) isolated from renal cortex of spontaneously hypertensive rats (SHR) and their normotensive control, Wistar Kyoto (WKY) rats have been studied. The activity of Na +-dependent D-glucose transport has been also determined. A significant increase in total phospholipids and free cholesterol were observed in renal BBMVs from SHR, which led to a decrease in the ratio of phospholipid-to-free cholesterol in these hypertensive rats. A reduction in the content of phosphatidylcholine and an increase in the percentage of sphingomyelin were observed in SHR. As a consequence, a diminished ratio of phosphatidylcholine-to-sphingomyelin was present in kidneys from SHR. The content of phosphatidylserine and phosphatidylinositol was reduced in SHR. However, the levels of phosphatidylethanolamine, cardiolipin, and lysophosphatidylethanolamine were increased in hypertensive animals. An increase in the level of saturated fatty acids, together with a decrease in the level of unsaturated fatty acids was responsible for the lower ratio of unsaturated-to-saturated fatty acids found in kidneys from SHR. The ratio of linoleic-to-arachidonic acid was increased in SHR when compared to WKY rats, as a result of the observed decrease in the content of arachidonic acid in hypertensive rats. Studies of diphenylhexatriene fluorescence polarization indicated no changes in the fluidity between BBMVs from both experimental rats, which did not correlate to the observed changes in the lipid composition of these membranes. These modifications in brush-border membrane lipid composition were accompanied by changes in the Na +-dependent d-glucose transport through renal BBMVs from SHR.

Osmotic Water Permeability of Vacuolar and Plasma Membranes Isolated from Maize Roots

The method of stopped flow was used to follow the changes in light scattering by the vesicles of plasmalemma and tonoplast isolated from maize (Zea maysL.) roots and treated by osmotic pressure. In both membrane preparations, the rate of the process depended on the osmotic gradient and was described with the simple exponential function. The rate constants derived from these functions were the following: the coefficient of water permeability in the tonoplast (P= 165 ± 7 μm/s) exceeded by an order of magnitude the corresponding index for plasmalemma (11 ± 2 μm/s). The presence of HgCl2(1.6 nmol/μg membrane protein) decreased the tonoplast water permeability by 80%. Microviscosity studies of the hydrocarbon zone in the isolated membranes by using a fluorescent diphenylhexatriene probe demonstrated that the two membranes do not differ in the phase state of their lipid bilayer. The authors conclude that the observed difference in water permeability does not depend on the state of the lipid phase and probably reflects the dissimilar functional activity of plasmalemma and tonoplast aquaporins.

Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity

Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5°C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.

Chronic changes in plasma triglyceride levels do modify platelet membrane microviscosity in rats

Lipid metabolism disorders were proposed to mediate numerous cell membrane alterations in various forms of hypertension. Elevated plasma triglycerides were found to be associated with changes in membrane structure and function related to altered microviscosity in particular domains of the cell membrane. The aim of our study was to determine if an abnormal triglyceride metabolism might play a causal role in these alterations of membrane dynamics. Using genetically hypertensive rats of the Prague hereditary hypertriglyceridemic (HTG) strain we investigated whether the elevation of circulating triglycerides induced by high fructose intake and/or their lowering by chronic gemfibrozil treatment (for 10 weeks starting at the age of 6 weeks) are followed by reciprocal changes in membrane microviscosity. Two different fluorescent probes exploring either the outer membrane leaflet (TMA-DPH anisotropy) or the membrane lipid core (DPH anisotropy) were used in platelets of HTG rats. DPH (diphenylhexatriene) fluorescence anisotropy was decreased in platelets of fructose-treated HTG animals with highly elevated plasma triglyceride levels, whereas it was increased in gemfibrozil-treated HTG rats in which triglyceride levels were almost normalized. On the contrary, TMA-DPH (trimethylamino-diphenylhexatriene) anisotropy was not substantially altered in platelets from HTG rats by the above modifications of circulating triglycerides. No changes of plasma cholesterol or blood pressure were associated with the triglyceride-dependent modifications of membrane core microviscosity. Our interventional study demonstrates a major causal role of circulating triglycerides in the control of the microviscosity of membrane lipid core.

Effect of long chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine on the interaction with recombinant high density lipoprotein apolipoprotein A-I

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.

113Cd-, 31P-NMR and fluorescence polarization studies of cadmium(II) interactions with phospholipids in model membranes

Cadmium(II) interactions with multilamellar vesicles of dimyristoyl (DM)- and dipalmitoyl (DP)-phosphatidylcholine (PC), -phosphatidylserine (PS), -phosphatidic acid (PA), -phosphatidylglycerol (PG) and -phosphatidylethanolamine (PE) have been investigated both from the metal and the membrane viewpoints, respectively, by solution 113Cd-NMR and diphenylhexatriene fluorescence polarization coupled with solid-state 31P-NMR. Results can be summarized as follows. (1) Strong cadmium binding to membrane phospholipids results in a decrease of the free Cd(II) 113Cd-NMR isotropic signal and because of slow exchange, in the NMR time scale, between free and bound cadmium pools, the lipid/water partition coefficients, K 1W of the Cd(II) species can be determined in the lamellar gel (fluid) phase. It is found K 1W(DMPC) ≈ K 1W(EggPE) ≈ 2 ± 2 (2±2); K 1W(DMPA) = 392±20 (505±25); K 1W(DMPG) = 428 ± 21 (352±17); K 1W(DMPS) = 544 ± 27 (672±34). Cadmium interactions with membrane phospholipids are therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. (2) The presence of Cd(II) stabilizes the gel phases of PG, PA and PS lipids and leads to suppression of the main phase transition for PA and PS. These effects are reduced upon increasing salinity to 0.5 M Cl − and abolished at 1.8 M Cl −, Cd(II) being removed from the membranes due to formation of soluble CdCl n species. Moving the pH from 7 to 6 also decreases Cd(II) binding to PA, because of surface charge reduction. (3) Cadmium promotes the formation of isotropic 31P-NMR lines with PG systems and of a hexagonal phase on egg PE bilayers at 24°C, suggesting dramatic membrane reorganization. Properties of cadmium and calcium interacting with phospholipid model membranes are compared, and the potential roles of these interactions in the molecular mechanisms of cadmium uptake and toxicity in cells are discussed.

Removal of dissolved O 2 by a fluid membrane of Fomblin Y (perfluoro polyether): fluorescence of diphenylhexatriene in Fomblin Y

O 2 can be gently removed from solutions by contact with a “bulk fluid membrane” of the viscous and nearly inert perfluoro polyether Fomblin Y. A volume of Fomblin dissolves ∼20 times more O 2 than an equal volume of water. Hence, when a volume of aqueous solution which was in equilibrium with air is enclosed with an equal volume of Fomblin which had been flushed with argon, oxygen would diffuse into the Fomblin, leaving in the water only 5% of the oxygen that was there. When the Fomblin is stirred, diffusion is rather rapid. The residue can be removed either by placing an oxygen scavenger on the other side of the Fomblin or by flowing a trickle of deoxygenated Fomblin through the sample. Diphenylhexatriene, a fluorescent probe of cell membranes, can be dissolved in Fomblin Y and has a fluorescence lifetimes extending from 12 ns in O 2 saturation to 30 ns (!) in the absence of oxygen. Stern–Volmer plots calibrated against a Clark electrode validate this system for oximetry. A general purpose anaerobic cuvette for fluorescence spectroscopy, containing the sample solution, a Fomblin compartment and the oxygen scavenger Na-dithionite is demonstrated.

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.

Destruction of membranes by merocyanine 540

The effect of a photosensitizer merocyanine 540 (MC 540) on the bilayer structure of model phospholipid membranes was studied using diphenylhexatriene (DPH) fluorescence anisotropy and light-scattering techniques. A pronounced reduction of DPH anisotropy decay within 60 min of DPH incubation was induced by the presence of MC 540 in lecithin membranes. With increasing MC 540 concentrations the original fluorescence peak of DPH in liposomes at 429 nm became less distinguishable and a new fluorescence peak at 403 nm was created. The presence of MC 540 induced also a concentration dependent decrease of the scattered light intensity. The observed changes could be interpreted in terms of disturbing the bilayer structure in the presence of MC 540.

Methyl mercury interactions with phospholipid membranes as reported by fluorescence, 31P and 199Hg NMR

Methylmercury (CH 3Hg(II)) interactions with multilamellar vesicles of dimyristoyl(DM)- and dipalmitoyl(DP)-phosphatidylcholine (PC), -phosphatidic acid (PA), -phosphatidylglycerol (PG), -phosphatidylserine (PS) and -phosphatidylethanolamine (PE) have been investigated from the metal viewpoint by solution 199Hg-NMR and from the membrane side by diphenylhexatriene fluorescence polarization and solid state 31P-NMR. Results can be summarized as follows: (1) CH 3Hg(II) strong binding to membranes results in a progressive decrease of the free CH 3HgOH 199Hg-NMR isotropic signal and because of a slow exchange, in the NMR time scale, between free and bound methylmercury pools the lipid/water partition coefficients, K lw, of the CH 3HgOH species can be determined in the lamellar gel (fluid) phase. It is found: K lw(DMPC)≈2±2 (2±2); K lw(DMPE)≈7±3 (16±3); K lw(DMPG)=170±10 (110±10); K lw(DMPS)=930±50 (1250±60); K lw(DMPA)=1250±60 (300±20). CH 3Hg(II) interactions with membrane phospholipids are therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. (2) The presence of CH 3HgOH stabilizes the PG gel phase and destabilizes that of PS. No effect is observed on PC, PA and PE thermotropism. (3) methylmercury promotes the formation of isotropic 31P-NMR lines with PG, PA and PE systems suggesting the presence of non-bilayer phases and hence membrane reorganization. The above effects are compared to those of inorganic mercury Hg(II) and discussed in the context of cell toxicity.

EFFECTS OF p-CHLOROTOLUENE (PCT) ON RAT LUNG AND LIVER BENZO[a]PYRENE METABOLISM AND MICROSOMAL MEMBRANE STRUCTURE AND FUNCTION

Treatment of rats with p-chlorotoluene (PCT, 1 g/kg, ip) resulted in peak PCT blood and lung concentrations at 4 h, which declined to very low levels at 12 h. The concentration of PCT in the liver attained its highest value at 1 h and also declined to low levels at 12 h. In the dose-response study, PCT significantly decreased hepatic and pulmonary AHH activities at 0.5 g/kg, 1 h. Maximum inhibition was attained at 1 g/kg, 1 h, and further increase in the dose did not enhance the enzyme inhibition. In the time-course investigation, PCT (1 g/kg) maximally inhibited hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities at 1 h and the decrease in enzyme activity was sustained through 12 h. Administration of PCT (1 g/kg, 1 h) also markedly decreased pulmonary cytochrome P-450 content, while hepatic cytochrome P-450 content was only slightly reduced. The partial decrease in cytochrome P450 content indicated altered levels of the P-450 isozymes, which may have profound effects on the metabolic disposition of benzo[a]pyrene [BaP]. BaP is regioselectively metabolized by two major isoforms of P-450 to toxic dihydrodiols and nontoxic phenol derivatives and there is a balance between these two metabolite groups. PCT (1 g/kg, 1 h) significantly inhibited the phenolic 3-OH BaP formation in both lung (52%) and liver (56%). The formations of BaP 7,8-dihydrodiol (146%) and 9,10-dihydrodiol (90%) were significantly elevated in the lung. The toxication to detoxication ratios were significantly elevated in both organs. Total quinone formation was markedly enhanced in the liver. Since PCT inhibited phenolic metabolite formation and increased dihydrodiol production, the activities of the isozymes that are responsible for their formations were determined. PCT (1 g/kg, 1 h) significantly inhibited cytochrome P-4502B1 in the lung (50%) and 2B1/2B2 in the liver (40%), while cytochrome P-4501A activity was not altered in either lung or liver. PCT increased phospholipid (PL) levels (45%) and conjugated diene (CD) formation (58%) in lung but not in liver, while membrane fluidity was increased [phospholipid/cholesterol (PL/CL) ratio; diphenylhexatriene (DPH) and trimethylammonium DPH (TMA-DPH) fluorescence polarization] in both organs. There was no apparent relationship between these membrane changes and alterations in MFO activity. Taken together the results suggest that PCT is capable of nonselectively inactivating the hepatic and pulmonary isozymes of P-450 that are responsible for the detoxication of BaP. The observed shift in the metabolism of BaP toward potentially more toxic metabolites suggests that concurrent exposure to BaP and PCT may result in greater toxicity, compared to exposure to BaP alone.

Diphenylhexatriene (DPH)-Labeled Lipids as a Potential Tool for Studies on Lipid Peroxidation in Monolayer Films

Using the fluorescence of diphenylhexatriene (DPH), lipid peroxidation in monomolecular films of phospholipids has been monitored dynamically to elucidate the efficacy of such a probe and to elucidate the effects of molecular organization on such peroxidation processes. Behavior in well-controlled model systems may be used to obtain insight into oxidative processes in complex biological systems. Mixed monolayers of hexadecanoyl-DPH-phosphatidylcholine (HDPH-PC) and diarachidonoyl-PC (DAA-PC) 1/10 (mol/mol) were prepared on a Langmuir trough. With pure water as subphase, and under an atmosphere of N 2/O 2 (3/1, v/v), DPH fluorescence remained stable over several hours, even under continuous illumination. However, over copper sulfate solution, rapid decay in the fluorescence intensity was observed and correlated with both the copper ion concentration and film pressure. No change in fluorescence was observed in absence of subphase copper ions or under an N 2 atmosphere. Substitution of dioleoyl phosphatidylcholine for DAA-PC in systems above gave no decrease in fluorescence intensity. This lipid alone is not susceptible to free radical chain reaction. Absorption spectra from HDPH-PC/DAA-PC monolayers confirm that DPH is actually destroyed during film oxidation. We thus propose this system as a new experimental model for studies on lipid peroxidation in organized systems. Copyright © 1997 Elsevier Science Inc.

Combined Fluorescence and NMR Studies of Reversed HPLC Stationary Phases with Different Ligand Chain Lengths

Alkyl chain bonded “reversed” HPLC phases consisting of 6 to 30 carbon atoms are investigated by fluorescence spectroscopy, steady-state and time-resolved fluorescence anisotropy, and solid-state NMR spectroscopy. The structure and dynamics of the interphase formed by alkyl chains and liquid phase penetrating each other are studied as a function of alkyl chain length. Increasing alkyl chain lengths lead to enhanced partitioning of the fluorescent probe diphenylhexatriene (DPH) into the interphase, as monitored by fluorescence decay curves. The concomitant spectral red shift of DPH fluorescence excitation maxima is evidence of increased interphase polarizability. Time-resolved fluorescence anisotropy measurements reveal that the motion of the probe molecule in the interphase is “wobble in cone”-like. Cone angles θ and rotational correlation times τR change from θ = 63° and τR = 0.75 ns in C6 phases to θ = 42° and τR = 1.50 ns in C30 phases, thus indicating decreasing probe mobility with increasing ligand length. This interpretation is supported by 13C CP/MAS NMR spectra, which show reduced contributions of alkyl chain gauche conformations, i.e., enhanced interphase order, in phases with long alkyl chains and high surface coverage. A concomitant increase in the line-widths of 1H MAS NMR peaks indicates reduced mobility of the longer chains. The spectroscopic observations are consistent with the results of HPLC separations, where enhanced shape selectivity is found with increasing ligand length, rod-shaped molecules like DPH showing the greatest increase in retention time.

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.

Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene

The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than α-tocopherol or α-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings.

Improved Red Blood Cell Preservation Correlates With Decreased Loss of Bands 3, 4.1, Acetylcholinestrase, and Lipids in Microvesicles

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.

In vitro regulation of low-density lipoprotein receptor interaction by fatty acids

Low-density lipoprotein (LDL) receptor binding is the initial step in receptor-mediated clearance. Dietary fat composition is known to affect LDL clearance, but the mechanism of the effect is unknown. We have examined the effects of altered membrane fatty acid composition, as might occur when specific dietary fats are consumed, on LDL binding using a Chinese hamster ovary (CHO) line that constitutively expresses the human LDL receptor. Binding of pooled human LDL to its receptor was compared in cells enriched with various fatty acids. Binding affinity was greater (lower K d ) for cells grown in 16:0-, 18:0-, or 18:1-enriched media than for those grown in 18:2 ( P < .0001). The apparent receptor number (Bmax) was lower for cells enriched in saturated fatty acids and 18:1. Fluidity was assessed by measuring diphenylhexatriene (DPH) fluorescence anisotropy ( r s ). Cells enriched in 18:1 or 18:2 were the most fluid ( P < .003). The correlation between binding and fluidity ( r = .24, P = .27) was weak and did not appear to explain the effects of fatty acid modification on LDL receptor binding. Thus, it appears that cellular enrichment in 16:0, 18:0, and 18:1 increases binding affinity by affecting properties other than membrane fluidity. Changes in Bmax may also contribute to the observed differences in LDL binding.